Kinases are the most intensively screened class of enzymes being targeted for therapeutic intervention because of their central role in regulating the function of other biomolecules. Strategies for developing selective kinase inhibitors are evolving rapidly, creating a demand for more flexible methods for measuring kinase activity in high throughput screening campaigns. Specifically, generic assays that can accommodate ATP concentrations spanning the physiological range are badly needed. Under separate SBIR funding, BellBrook Labs developed and commercialized a novel kinase enzyme assay method called Transcreener(R) that relies on selective immunodetection of ADP, thus enables detection of any kinase regardless of the acceptor substrate. The assay has been widely adopted by pharmaceutical industry, and licensed by leading drug discovery service providers, providing a significant revenue stream to BellBook. Despite its technical advantages and commercial success, significant drawbacks of the method include the need to adjust antibody concentration to accommodate different ADP levels, and a negative signal in response to ADP because of the competitive nature of the assay. To overcome these limitations, we will collaborate with Dr. Bruce Hammock at University of California, Davis to develop phage-borne peptides that bind specifically to the ADP-antibody immune complex. The use of these phage-peptides in a trivalent immune complex is expected to increase the selectivity for ADP detection vs. ATP, thereby overcoming the major limitation with the Transcreener platform. In addition, the resulting noncompetitive immunoassay, called a Phage Anti-Immune Complex (PHAIA), will generate a signal that is directly proportional to product formation in either homogenous (e.g., TR-FRET), or solid phase (e.g., ELISA) formats, thus greatly extending the flexibility and applicability of the method. PUBLIC HEALTH RELEVANCE: Kinases are one of the largest families of proteins in humans, and their malfunction is involved in many types of diseases, especially cancers, inflammation-related diseases. This proposal seeks to produce improved tools for identifying selective kinase inhibitors that can be used as drugs to treat kinase-related diseases.